This invention relates to methods for producing xcex1(2-3) sialyloligosaccharides in a dairy source or cheese processing waste stream by contacting the dairy source or cheese processing waste stream with a catalytic amount of at least one xcex1(2-3) trans-sialidase. In preferred embodiments, the methods of the invention are applied to produce xcex1(2-3) sialyllactose in a dairy source or cheese processing waste stream. Methods for isolating the xcex1(2-3) sialyloligosaccharides produced according to the methods of the invention are also provided. The invention additionally relates to a method for producing xcex1(2-3) sialyllactose in milk using a transgenic mammal containing an xcex1(2-3) trans-sialidase encoding sequence operably linked to a regulatory sequence of a gene expressed in mammary tissue.
2.1. Sialyloligosaccharides in Cheese Waste Streams
Whey is a major by-product of cheese manufacturing, which, for environmental reasons, presents a difficult waste disposal problem. In the United States alone, fluid whey is being produced at a rate of about 62.6 billion pounds annually. Whey is typically composed of about 5 wt. % lactose, 1 wt. % protein and about 0.5 wt. % salts, where the balance of the mixture is water. A major effort by many cheese making countries is presently underway to develop uses for this commodity, which formerly was considered a cheese processing waste product.
Although the protein concentrate obtained by ultrafiltration of whey has become a valuable commodity in the food industry and has found applications in animal feed, fertilizer, fermentation, and food filler, the majority of the resulting lactose-rich ultrafiltered permeate is still considered a disposable fraction.
Recently, several sialyloligosaccharides have been found to have valuable application as pharmaceutics. See, e.g. U.S. Pat. No. 5,270,462 to Shimatani et al. Sialyllactose has been shown to neutralize enterotoxins of various pathogenic microbes including Escherichia coli, Vibrio cholerae and Salmonella. See, e.g. U.S. Pat. No. 5,330,975 to Hiroko et al. It has also been shown that xcex1(2-3) sialyllactose (xcex1-Neu5Ac-(2-3)-Gal-xcex2-(1-4)-Glc) interferes with colonization of Helicobacter pylori and thereby prevents or inhibits gastric and duodenal ulcers. See e.g. U.S. Pat. No. 5,514,660 to Zopf et al. Sialyllactose has additionally been proposed to inhibit immune complex formation by disrupting occupancy of the Fc carbohydrate binding site on IgG and to be useful in treating arthritis. See, e.g. U.S. Pat. No. 5,164,374 to Rademacher et al.
To date, commercially available sialyloligosaccharides have been very expensive due to their low quantity in natural sources. For example, xcex1(2-3) sialyllactose and xcex1(2-6) sialyllactose isolated from bovine colostrum, is sold for $75.60 and $83.30 per milligram, respectively (Sigma Chemical Company, 1997).
A focused effort has been directed toward harvesting sialyloligosaccharides from the vast supply of whey made available as a cheese processing waste product. Processes for isolating sialyloligosaccharides have utilized such techniques as ultrafiltration, ion-exchange resins and phase partition chemistry. U.S. Pat. No. 4,001,198 to Thomas and U.S. Pat. No. 4,202,909 to Pederson; U.S. Pat. No. 4,547,386 to Chambers et al.; U.S. Pat. No. 4,617,861 to Armstrong; U.S. Pat. Nos. 4,971,701 and 4,855,056 to Harju et al.; U.S. Pat. No. 4,968,521 to McInychyn; U.S. Pat. No. 4,543,261 to Harmon et al.; U.S. Pat. Nos. 5,118,516 and 5,270,462 to Shimatani; JP Kokai 01-168,693; JP Kokai 03-143,351; JP Kokai 59-184,197; JP Kokoku 40-1234; JP Kokai 63-284,199 and Japanese Patent Publication No. 21234/1965, each of which is herein incorporated by reference in its entirety. Yields of up to 6 grams of xcex1(2-3) sialyllactose sialyloligosaccharide per kilogram of cheese processing waste stream have been reported. U.S. Pat. No. 5,575,916 to Brian et al. which is herein incorporated by reference in its entirety.
2.2. Sialidases and Sialyltransferases
Sialic acids are 9-carbon carboxylated sugars which generally occur as the terminal monosaccharides in oligosaccharide chains. In mammalian cells, sialic acids are most frequently linked to xcex2-galactose with an xcex1(2-3) linkage, and to N-acetylglucosamine and N-acetylgalactosamine with an xcex1(2-6) linkage. Cross et al., 1993, Annu. Rev. Microbiol. 47:385-411.
Sialidases catalyze the removal of sialic acid residues from the oligosaccharide chain. Due to the wide variety of substitutions which may occur at various carbons of the sialic acid molecules, there are at least 39 different species of sialic acids. Colli, W., 1993, FASEB J. 7:1257-1264. Generally, sialidases exhibit substrate specificity for specific forms of sialic acid linkages. Viral sialidases cleave xcex1(2-3) glycosidic bonds more efficiently than xcex1(2-6) bonds, but bacterial sialidases are not as specific. Cross et al., 1993, Annu. Rev. Microbiol. 47:385-411 (citing Corfield et al. 1982, Sialic Acids: Chemistry, Metabolism and Function, Vol. 10, New York: Springer-Verlag, pp. 195-261). At low enzyme concentrations, bacterial sialidases exhibit a preference for cleaving xcex1(2-3) or xcex1(2-6) glycosidic bonds. Cross et al., 1993, Annu. Rev. Microbiol. 47:385-411.
CMP-sialyltransferases catalyze the transfer of cytidine monophosphate-sialic acid (CMP-sialic acid) residues to acceptor molecules. Although many sialidases exhibit at least some substrate specificity, CMP-sialyltransferases act on specific substrates. Mammalian CMP-sialyltransferases are generally found in the Golgi, however, there is evidence that there may be cell-surface associated CMP-sialyltransferases as well. Cross et al., 1993, Annu. Rev. Microbiol. 47:385-411 (citing Roth et al., 1971, J. Cell Biol. 51:536-547; Shur, 1991, Glycobiology 1:563-575; Yogeeswaran et al., 1974, Biochem. Biophys. Res. Commun. 59:591-599).
2.3. Trypanosoma Cruzi xcex1(2-3)-Trans-Sialidase
Trypanosoma cruzi (Order Kinetoplastida) is the intracellular parasite responsible for Chagas disease, throughout Iberoamerican countries. Chagas disease primarily affects nerve and muscle cells. One serious manifestation of Chagas disease is a chronic progressive fibrotic myocarditis. Colli, 1993, FASEB J. 7:1257-1264. Approximately 16-18 million people are infected with T. cruzi. Colli, 1993, FASEB J. 7:1257-1264.
T. cruzi invades a broad range of host cells, and a considerable amount of research has focused on the surface molecules in order to determine which molecules may be involved in parasite/host interaction. Colli, 1993, FASEB J. 7:1257-1264. One surface molecule which has generated a great deal of interest is the xcex1(2-3)-trans-sialidase. This molecule has the capability of catalyzing both the removal of sialic acid from a donor saccharide-containing molecule (sialidase activity) and catalyzing the transfer of the sialic acid to an acceptor saccharide-containing molecule (trans-sialidase activity). Schenkman et al., 1992, J. Exp. Med. 175:567-575. The gene encoding T. cruzi trans-sialidase has been cloned and characterized at the molecular level.
The T. cruzi xcex1(2-3) trans-sialidase catalyzes the transfer of sialic acid from a donor terminal xcex2-galactosyl sialoglycoconjugate to a terminal xcex2-galactose on an acceptor molecule. Colli, W., 1993, FASEB J. 7:1257-1264. T. cruzi xcex1(2-3) trans-sialidase does not use CMP-sialic acid as a substrate and prefers sialyl xcex1(2-3)-linked to xcex2-galactosyl residues as sialic acid donor molecules over sialyl xcex1(2-6)-, xcex1(2-8)-, and xcex1(2-9)-linked sialic acids. Schenkman et al., 1994, Annu. Rev. Microbiol. 48:499-523. Furthermore, T. cruzi xcex1(2-3) trans-sialidase cannot use free sialic acid as a substrate. Vandekerckhove et al. 1992, Glycobiology 2:541-548. The T. cruzi xcex1(2-3) trans-sialidase has a broad pH optimum centered at 7.0. Cross et al., 1993, Annu Rev. Microbiol. 47:385-411.
More detailed analysis of the xcex1(2-3) trans-sialidase has revealed that the amino-terminal portion of the protein is responsible for the xcex1(2-3) trans-sialidase activity. Campetella et al., 1994, Mol. Biochem. Parasitol. 64:337-340; Schenkman et al., 1994, J. Biol. Chem. 269:7970-7975. It has also been determined that there are at least two critical amino acid residues: Tyr342 and Pro231 of the xcex1(2-3) trans-sialidase appear to be required for full xcex1(2-3) trans-sialidase activity. Cremona et al., 1995, Gene 160:123-25. The importance of Tyr342 is demonstrated by the fact that naturally occurring variants of the T. cruzi xcex1(2-3) trans-sialidase which have a Tyr342xe2x86x92His substitution, lack xcex1(2-3) trans-sialidase activity. Uemura et al., 1992, EMBO J. 11:3837-3844.
Trans-sialidase activity has also been discovered in Trypanosoma brucei, the causative agent of African Sleeping Sickness, Endotrypanum spp. and in Pneumocystis carinii. Like the T. cruzi xcex1(2-3) trans-sialidase, the T. brucei trans-sialidase has a pH optimum of 7.0. However, unlike the T. cruzi trans-sialidase, which is expressed during the trypomastigote stage, the T. brucei trans-sialidase appears to be expressed only during the procyclic stage of the parasite life cycle, when the parasite resides in the midgut of its insect vector (Glossina spp., the xe2x80x9ctsetse flyxe2x80x9d). Cross et al., 1993, Annu Rev. Microbiol. 47:385-411.
2.4. Sialyllactose Production
A variety of methods for enzymatically producing sialylated oligosaccharides have been described.
U.S. Pat. No. 5,374,541 to Wong et al., describes a method for producing sialyloligosaccharides. According to this method, xcex2-galactosidase is used to form xcex2-galactosyl glycosides in the presence of CMP-sialic acid and xcex1(2-3)- or xcex1(2-6)-CMP-sialyltransferases to form sialylated oligosaccharides. This method does not use xcex1(2-3) trans-sialidase.
U.S. Pat. No. 5,409,817 to Ito et al., discloses a three enzyme process for producing xcex1(2-3) sialylgalactosides. According to this process, CMP-sialyltransferases transfer sialic acid from CMP-sialic acid to acceptor molecules, these acceptor molecules become donor molecules for Trypanosoma cruzi xcex1(2-3) trans-sialidase, and CMP-sialic acid is regenerated in the system through the action of CMP-sialic acid synthetase and added free sialic acid.
The process described in U.S. Pat. No. 5,409,817 to Ito et al. specifically requires the addition of free sialic acid. The free sialic acid is converted to CMP-sialic acid by CMP-sialic acid synthetase, and the sialic acid moiety is transferred to an acceptor molecule by CMP-sialyltransferase. according to the disclosure of Ito et al., the formation of these sialylated acceptor molecules is required to drive the xcex1(2-3) trans-sialidase reaction forward.
In addition to free sialic acid, the method of Ito et al., also requires the presence of three enzymes including CMP-sialic acid synthetase and CMP-sialyltransferase. Further, dairy sources and cheese processing waste streams do not contain CMP-sialic acid synthetase.
2.5. Expression of Transgenes in Milk
Numerous foreign proteins have successfully been transgenically expressed in the milk of livestock. Most of this work has focused on the expression of proteins which are foreign to the mammary gland. Colman, A., 1996, Am. J. Clin. Nutr. 63:639S-645S. To date, milk specific expression of transgenic livestock has been achieved through operably linking regulatory sequences of milk-specific protein genes to the target protein-encoding gene sequence, microinjecting these genetic constructs into the pronuclei of fertilized embryos, and implanting the embryos into recipient females. See e.g. Wright et al., 1991, Biotechnology (NY) 9:830-834; Carver et al., 1993, Biotechnology (NY) 11:1263-1270; Paterson et al., 1994, Appl. Microbiol. Biotechnol. 40:691-698. Proteins that have been successfully expressed in the milk of transgenic animals, include: xcex11-antitrypsin (Wright et al., 1991, Biotechnology (NY) 9:830-834; Carver et al., 1993, Biotechnology (NY) 11:1263-1270); Factor IX (Clark et al., 1989, Biotechnology (NY) 7:487-492); protein C (Velander et al., 1992, Proc. Natl. Acad. Sci. USA, 89:12003-12007); tissue plasminogen activator (Ebert et al., 1991, Biotechology (NY) 9:835-838); and fibrinogen. While most of these transgenes express proteins that supplement the composition of milk, very few, if any of the expressed proteins interact directly with the components of milk to alter the natural milk composition. There is a need for methods providing for the large scale production of xcex1(2-3) sialyloligosaccharides, such as xcex1(2-3) sialyllactose, which have commercial and/or therapeutic valve.
The present invention greatly advances the field of commercial production of sialyloligosaccharides by providing methods for producing sialyloligosaccharides in situ in dairy sources and cheese processing waste streams. The methods of the invention have particular applications in producing xcex1(2-3) sialyllactose in a dairy source prior to, during, or after processing of the dairy source during the cheese manufacturing process, thereby greatly increasing the recoverable yield of xcex1(2-3) sialyllactose from the dairy source.
Dairy sources and cheese processing waste streams are known to contain high concentrations of lactose and numerous xcex1(2-3) sialosides, such as, for example, xcexa casein, and the gangliosides. Applicants are the first to provide a method for producing xcex1(2-3) sialyllactose in a dairy source or a cheese processing waste stream. More specifically, the method of the present invention uses the catalytic activity of xcex1(2-3) trans-sialidases to exploit the high concentrations of lactose and xcex1(2-3) sialosides which naturally occur in dairy sources, to drive the enzymatic synthesis of xcex1(2-3) sialyllactose. This catalytic activity does not require the presence of CMP-sialic acid synthetase, CMP-sialyltransferase and/or free siallic acid to drive the sialylation of xcex1(2-3) sialyllactose and other xcex1(2-3) sialyloligosaccharides.
Accordingly, the invention provides a novel method for producing xcex1(2-3) sialyloligosaccharides, and specifically, xcex1(2-3) sialyllactose (xcex1-Neu5Ac-(2-3)-Gal-xcex2-(1-4)-Glc), in a dairy source or cheese processing waste stream by catalyzing the sialidation of lactose (Gal-xcex2-(1-4)-Glc). In specific embodiments, the method of the invention is applied to the dairy source prior to or during processing. In another specific embodiment, the method of the present invention is applied after processing of the dairy source (e.g. to a cheese processing waste stream).
The present invention provides a method for producing sialyloligosaccharides in a dairy source. This method comprises contacting a catalytic amount of least one xcex1(2-3) trans-sialidase with a dairy source to form a dairy/trans-sialidase mixture and incubating the dairy/trans-sialidase mixture under conditions suitable for xcex1(2-3) trans-sialidase activity. xcex1(2-3) sialyloligosaccharides produced according to this method are additionally encompassed by the present invention. The invention also provides for recovery of the sialyloligosaccharides contained in the incubated dairy/trans-sialidase mixture or alternatively, in compositions formed after processing of the incubated dairy/trans-sialidase mixture (e.g. a cheese processing waste stream), using techniques which include, but are not limited to, ultrafiltration, diafiltration, nanofiltration, electrodialysis, phase partitioning and ion exchange chromatography.
The present invention also provides a method for producing sialyloligosaccharides in a cheese processing waste stream. This method comprises contacting a catalytic amount of at least one xcex1(2-3) trans-sialidase with a cheese processing waste stream to form a waste stream/trans-sialidase mixture and incubating the waste stream/trans-sialidase mixture under conditions suitable for xcex1(2-3) trans-sialidase activity. xcex1(2-3) sialyloligosaccharides produced according to this method are additionally encompassed by the present invention. The invention also provides for recovery of the sialyloligosaccharides contained in the incubated dairy/trans-sialidase mixture using techniques which include, but are not limited to, ultrafiltration, diafiltration, nanofiltration, electrodialysis, phase partitioning and ion exchange chromatography.
The invention further provides a method for producing xcex1(2-3) sialyllactose. This method comprises contacting a catalytic amount of at least one xcex1(2-3) trans-sialidase with lactose and an xcex1(2-3) sialyloligosaccharide, in the absence of CMP-sialyltransferase, to form a mixture, and incubating this mixture under conditions suitable for xcex1(2-3) trans-sialidase activity. xcex1(2-3) sialyllactose produced according to this method are additionally encompassed by the present invention. The invention also provides for recovery of the sialyllactose contained in this incubated mixture using techniques which include, but are not limited to, ultrafiltration, diafiltration, nanofiltration, electrodialysis, phase partitioning and ion exchange chromatography.
The invention additionally provides a method of enriching for xcex1(2-3) sialyllactose in milk using transgenic mammals that express an xcex1(2-3) trans-sialidase transgene. According to this method, a transgene comprising an xcex1(2-3) trans-sialidase encoding sequence is operably linked to a regulatory sequence of a gene expressed in mammary tissue and this xcex1(2-3) trans-sialidase/regulatory sequence transgene is then introduced into the germline of a mammal to produce a transgenic mammal. The milk produced by a transgenic mammal demonstrating xcex1(2-3) trans-sialidase activity in mammary tissue, contains enriched xcex1(2-3) sialyllactose concentrations. The invention also provides for recovery of the sialyllactose contained in the milk produced by this transgenic mammal either before or after processing of the milk. Transgenic mammals containing an xcex1(2-3) trans-sialidase encoding sequence operably linked to a regulatory sequence of a gene expressed in mammary tissue are also provided by the invention. Significantly, a dairy source, cheese processing waste stream, and transgenic mammal can be used to produce enriched concentrations of xcex1(2-3) sialyllactose.
As used herein, xe2x80x9ctrans-sialidasexe2x80x9d refers to a compound that catalyzes the transfer of a sialic acid from one saccharide-containing molecule (e.g. oligosaccharide, polysaccharide, glycoprotein or glycolipid) to another saccharide-containing molecule and which does not require presence of free sialic acid, CMP-sialic acid, synthetase and/or CMP-sialyltransferase in the reaction mixture for its activity.
As used herein, xe2x80x9ctrans-sialidase activityxe2x80x9d refers to the catalytic reaction in which an enzyme catalyzes the removal of a sialic acid from one saccharide-containing molecule and the transfer of the sialic acid to another saccharide-containing molecule, covalently attaching the sialic acid to the acceptor molecule through a glycosidic bond.
As used herein, a xe2x80x9ccatalytic amountxe2x80x9d of xcex1(2-3) trans-sialidase enzyme refers to the quantity of enzyme sufficient to cause the transfer of a sialic acid from one saccharide-containing molecule to another saccharide-containing molecule.
As used herein, xe2x80x9cconditions suitable for trans-sialidase activityxe2x80x9d encompass appropriate conditions (e.g. temperature, pH and incubation time) sufficient to permit the enzymatic removal of a sialic acid from one saccharide-containing molecule and the transfer of the sialic acid to another saccharide-containing molecule.
As used herein, xe2x80x9cxcex1(2-3) sialyloligosaccharidesxe2x80x9d refer to sugars in which a sialic acid is covalently attached to the 3xe2x80x2 carbon of a xcex2-galactose moiety through a glycosidic bond. In the methods of the present invention, xcex1(2-3) sialyloligosaccharides encompass saccharides with any form of sialic acid covalently attached to the 3xe2x80x2-xcex2-galactose.
As used herein, xe2x80x9cdairy sourcexe2x80x9d refers to a product of lactation in a mammal, a substance made by the product, or a byproduct thereof. As used herein, xe2x80x9cdairy sourcexe2x80x9d includes, but is not limited to, milk, colostrum, a cheese processing mixture, and a composition simulating milk.
As used herein, a xe2x80x9ccheese processing mixturexe2x80x9d is a compilation of ingredients of dairy processing at any stage during dairy processing (e.g. pasteurization, fermentation, or cheese manufacture) other than the cheese processing waste stream.
As used herein, xe2x80x9ca composition simulating milkxe2x80x9d is a solution lacking one or more of CMP-sialyltransferase, CMP-synthetase and/or free sialic acid, but which contains at least xcex1(2-3) sialosides to act as donors for the trans-sialidase, lactose and, optionally, appropriate buffering agents to maximize the activity of the xcex1(2-3) trans-sialidase when it is added to the solution.
As used herein, xe2x80x9ccheese processing waste streamxe2x80x9d refers to a byproduct of cheese manufacture and includes, but is not limited to, whole whey, demineralized whey permeate, the regeneration stream from demineralized whey permeate, whey permeate, crystallized lactose, spray dried lactose, whey powder, edible lactose and lactose. Whey containing sialic acids, is a byproduct obtained when cheese or rennet casein is produced from milks such as cow milk, goat milk, and sheep milk. For example acid whey, is generated by separating the solids when skim milk is coagulated to form cottage cheese. Acid whey is characterized by a high lactic acid content. When cheese is prepared from whole milk, the remaining liquid is sweet whey, which can be further processed by evaporation to form dry whey powder. Sweet whey can also be dried, demineralized and evaporated to form demineralized whey permeate. Sweet whey can also be subjected to ultrafiltration to generate both a whey permeate and a whey protein concentrate. Whey permeate can be further processed by crystallizing lactose to form both lactose and a mother liquor. The mother liquor resulting from crystallizing lactose from a whey permeate is known in the art as xe2x80x9cDelac.xe2x80x9d
The xcex1(2-3) trans-sialidase used according to the method of the present invention encompasses Kinetoplastid trans-sialidases, trans-sialidases derived from Trypanosoma, Endotrypanum, and Pneumocystis, and includes trans-sialidases of Trypanosoma cruzi, Trypanosoma brucei, Endotrypanum spp. and Pneumocystis carinii. Trans-sialidases that may be used according to the method of the present invention are further defined infra in Section 5.1.